Visual information is recognized in such a manner that light transmitted into the cornea, which is a transparent tissue at the forefront of an eyeball, reaches the retina to excite the nerve cell of the retina, and an electric signal generated is transferred through the optic nerve to the visual cortex of the cerebrum. In order to obtain good visual acuity, it is necessary that the cornea is transparent. The transparency of the cornea is maintained by retaining a constant moisture content with the pumping function and barrier function of a corneal endothelial cell.
The cornea is a transparent tissue which is positioned in front of the eyeball and the structure of cornea is mainly composed of three-layers known as corneal epithelial cell layer, corneal stromal layer and corneal endothelial cell layer. The corneal endothelial cell layer is a single cell layer existing in a corneal deep part, has the barrier function and the pumping function, and plays a role in maintaining the transparency of the cornea by retaining a constant corneal moisture amount. In addition, it is known that even if the corneal endothelial cells are damaged, they do not proliferate in a living body, and also it is known that a serious visual disorder is generated by damaging corneal endothelial cells with trauma, a disease or the like to decrease their number.
Human corneal endothelial cells exist at a density of about 3000 cells per 1 square millimeter at birth, but once the corneal endothelial cells are damaged, they have no ability to regenerate themselves. In endothelial corneal dystrophy or bullous keratopathy which is generated by dysfunction of the corneal endothelium due to a variety of causes, the cornea becomes opaque due to edema, which leads to remarkable reduction in the visible acuity. Currently, penetrating keratoplasty for transplanting the whole three-layered structure of epithelium, stroma and endothelium of the cornea is performed for bullous keratopathy. However, donation of the cornea in Japan is insufficient, and the number of waiting patients for corneal transplantation is about 2600, but the number of corneal transplantation performed by using the cornea from a donor in Japan per year is about 1700.
In recent years, for the purpose of reducing the risk of immunological rejection or the risk of postoperative complications and obtaining better visual function, the idea of “parts transplantation” for transplanting a damaged tissue alone has been drawing the attention. Among the cornea transplantations, deep layer and superficial layer corneal transplantations in which the stromal tissue is transplanted, Descemet's Stripping Automated Endothelial Keratoplasty in which the corneal endothelial tissue is transplanted, and the like have been performed. In addition, cultured mucosal epithelial transplantation in which the corneal epithelium or the oral mucosa which has been cultured in vitro is transplanted in place of the corneal epithelium has been already applied clinically, and a method for transplanting the corneal endothelium which has been cultured in vitro similarly has been also studied.
It is known that a corneal endothelial cell is differentiated when cultured, and morphology changes to a fibroblast cell-like form. In addition, it is known that GPR49/LGR5 is expressed in a small intestine epithelial stem cell in a limited manner, and plays an important role. As a ligand of GPR49/LGR5, R-spondins are reported (Non-Patent Documents 1 to 4).
Non-Patent Document 5 describes that a stem cell-like cell exists in a corneal limbal epithelial cell, and GPR49/LGR5 can be a phenotype marker of a remaining human corneal limbal epithelial stem cell.
In Non-Patent Document 6, GPR49/LGR5 is exemplified as a stem cell marker. Non-Patent Document 6 describes that, in two cell populations of mouse corneal epithelial cells, GPR49/LGR5 and ABCG2 are highly expressed.
Non-Patent Document 7 describes GPR49/LGR5 as a stem cell marker.
Non-Patent Document 8 discloses that intestinal tract epithelial stem cell culturing is established.
Non-Patent Document 9 discloses a method of small intestinal culture and a large intestinal culture in a stable manner and for a long term. Non-Patent Document 9 describes that culture growth is markedly stimulated by a fusion protein (RSpol-Fc) between R-spondin 1 and immunoglobulin Fe.
Previously, it has been considered that corneal endothelial cells do not proliferate in vivo, but by molecular biological or cellular biological study in recent years, it has been reported that a cell group very rich in proliferation ability also exists in the corneal endothelial cell layer. Schimmelpfenning et al. revealed that the endothelial cell density is increased more at a peripheral part of the human cornea than at a central part, and proposed a possibility that the proliferation of cells at a corneal peripheral part supplies cells to a corneal central part (Non-Patent Document 10).
In addition, using the rabbit cornea, Whikehart et al. confirmed that telomerase, which is observed to be highly expressed in a stem cell or a precursor cell, is highly expressed in an endothelial cell at a corneal peripheral part. Further, by an evaluation method of using BrdU as a cell proliferation marker, it is shown that a fast response is observed when an endothelial cell at a corneal peripheral part is damaged (Non-Patent Document 11). In addition, Yokoo et al. succeeded in collection of a precursor cell of a corneal endothelial cell from the adult human cornea using a sphere method which is a method for collecting a mesenchymal stem cell (Non-Patent Document 12). This cell expresses an undifferentiated cell marker, and when the ability to form a sphere was compared between corneal endothelial cells at a central part and a peripheral part, it was confirmed that the ability is high in a peripheral endothelial cell (Non-Patent Document 13). Furthermore, McGowan et al. reported that a large number of cells expressing an undifferentiated cell marker exist at a corneal peripheral part, and these cells are activated by being damaged (Non-Patent Document 14).
G protein-coupled receptor 49 (also referred to as “GPR49/LGR5” as used herein) is one kind of seven-transmembrane receptors similar to thyroid-stimulating hormone, follicle-stimulating hormone (FSH), or leuteinizing hormone (LH), and has a unique structure associated with an extracellular N-terminal domain including a leucine rich repeat (FIG. 1, Non-Patent Document 15). It has been reported that since GPR49/LGR5 is a target gene of Wnt-signaling pathway and Hedgehog-signaling pathway involved in oncogenesis, expression of GPR49/LGR5 is elevated by abnormality of these signaling pathways (Non-Patent Document 16, Non-Patent Document 17 and Non-Patent Document 18). Furthermore, since it was confirmed that specific expression of GPR49/LGR5 is seen in an intestinal tract epithelial stem cell (Non-Patent Document 8), GPR49/LGR5 has been drawing attention as a novel protein which is expressed in a stem cell-specific manner. Thereafter, it has been confirmed that expression of GPR49/LGR5 is elevated in a stem cell-specific manner also in a tissue such as hair follicle (Non-Patent Document 19) or stomach epithelium (Non-Patent Document 20), and a possibility that GPR49/LGR5 is involved in construction of stem cell niche and tissue formation has been also reported.